The Role Of Toll-Like Receptor 4 In Hypoxia-Reoxygenation-Induced Apoptosis In Rat Cardiac Myocytes And Effects Of Simvastatin On Them

Objective Myocardial ischemia and reperfusion injury(MI/RI)is one of the common injury during the treatment of coronary atherosclerotic heart disease(CAD),and it is closely related to apoptosis of cardiomyocyte.Evidence suggest that Toll-like receptor 4(TLR4)involved in the process of apoptosis in myocardium caused by MI/RI.However,the dynamic expressions of TLR4 and possible molecules regulated by TLR4 in cardiac myocyte subjected to hypoxia and reoxygenation remain unclear.Simvastatin possesses the anti-apoptotic effect,but few studies about the role of TLR4 and possible associated molecules in this cardioprotective effect.Therefore the present study is aimed to address the role of TLR4 in hypoxia-reoxygenation-induced damage in rat cardiac myocytes and the effects of simvastatin on them.Methods Established the cellular model with exposure of neonatal rat ventricular myocytes(NRVMs)to hypoxia-reoxygenation in vitro.NRVMs were isolated from one day-old neonatal Sprague Dawley rats.After culture for five days,the NRVMs were divided into control and hypoxia-reoxygenation group.The NRVMs in hypoxia-reoxygenation group were subjected to hypoxia for 4h alone and/or followed reoxygenation for another 2,4h respectively.The apoptotic NRVMs was determined with flow cytometry(FCM),and apoptosis marker cleaved Caspase-3 expression in NRVMs was assessed by Western Blot.The level of TLR4 m RNA was measured by quantitive real time polymerase chain reaction(q PCR)and TLR4 protein expression was assessed by Western Blot after exposure of NRVMs to hypoxia-reoxygenation.Then H9c2 cardiomyoblasts,used to simulate NRVMs,were divided into three groups:control group,hypoxia 4h-reoxygenation 4h(H4h/R4h)group,H4h/R4 h plus TAK-242 group.Changes of expression levels and phosphorylation of the p38 mitogen actived protein kinase(p38 MAPK),c-Jun N-terminal kinase(JNK)and protein kinase B(Akt)in cardiac cells caused by hypoxia-reoxygenation were all detected by Western Blot.Later,the H9c2 cardiomyoblasts were divide into the following groups:control group,H4h/R4 h group and H4h/R4 h plus simvastatin group.Finally,Western Blot was done to assessed the expressions of these molecules above.Results Exposure of cultured NRVMs to hypoxia-reoxygenation led to cellular apoptosis.Results from FCM and Western Blot have shown that apoptosis and expression of cleaved Caspase-3 in NRVMs significantly increased(P<0.05).Detection both with q PCR and with Western Blot revealed that TLR 4 m RNA and protein of NRVMs remarkly upregulated after exposure for hypoxia-reoxygenation.Subsequently,Western Blot assay showed that this stimulation induced the protein levels of p38 MAPK and the ratio of phosphorylated(p)-p38/total(t)-p38 MAPK significantly increased in H9c2 cells,as well as the expression of total c-Jun N-terminal kinase(JNK)and the ratio of p-JNK/t-JNK.But the molecular changes were reversed by the pretreatment of TLR4 antagonist TAK-242.The protein level of t-Akt increased slightly but the ratio of p-/t-Akt decreased markly in H4h/R4 h group,whereras the ratio of p-/t-Akt raised in the H4h/R4h+TAK-242 H9c2 cells(P<0.05).Compared to H4h/R4 h group,the expressions of TLR4 and cleaved Caspase-3were decreased along with an increase in simvastatin concentration.The levels of t-p38 MAPK,t-JNK and the ratio of p-p38/t-p38 MAPK and p-/t-Akt of H9c2 cardiomyoblasts in H4h/R4 h +10μM simvastatin group profoundly downregulated whereas the level of t-Akt and the ratio of p-/t-Akt upregulated(P<0.05).But there was no statistic difference of t-Akt levels between simvastatin pretreatment group and normal control group(P>0.05).Conclusion TLR4 involved in the apoptosis of rat cardiac myocyte induced by hypoxia-reoxygenation via regulating the expressions of p38 MAPK,JNK and Akt.Simvastatin pretreatment protect cardiac myocyte from damage through downregulation of TLR4 that influencing p38 MAPK,JNK and Akt.